The present invention is directed to a method for the isolation and detection of normal, benign hyperplastic, and cancerous prostate epithelial cells from semen, using a magnetic activated cell sorter (MACS).
In spite of improved treatments for certain forms of cancer, it is still a leading cause of death in the United States. Since the chance for complete remission of cancer is, in most cases, greatly enhanced by early diagnosis, it is very desirable that physicians be able to detect cancers before a substantial tumor develops. However, the development of methods that permit rapid and accurate detection of many forms of cancer continues to challenge the medical community. One such illustrative form of cancer is prostate cancer.
Prostate cancer is the most common cancer in men with an estimated 317,000 cases in 1996 in the United States. It is the second leading cause of death among men who die from neoplasia with an estimated 40,000 deaths per year. Prompt detection and treatment is needed to limit mortality caused by prostate cancer.
Screening tests (i.e., digital rectal examination and prostatic specific antigen levels) are widely used for early detection of potentially curable prostatic cancer, but an accurate cytologic or histologic assessment is necessary to confirm the proper and accurate diagnosis. A prostatic needle biopsy is specific, but invasive and has a significant false negative rate (Stroumbakis, N. et al., Urology, 49 (Suppl. 3A):113-118 (1997)). Furthermore, this procedure is associated with a significant level of morbidity, such as infection and bleeding.
When it metastasizes, prostatic cancer has a distinct predilection for bone and lymph nodes (Saitoh et al., Cancer, 54:3078-3084 (1984)). At the time of clinical diagnosis, as many as 25% of patients have bone metastasis demonstrable by radionuclide scans (Murphy, G. P., et al., J. Urol., 127:928-939 (1982)). Accurate clinical evaluation of nodal involvement has proven to be difficult. Imaging techniques such as computed tomography (xe2x80x9cCTxe2x80x9d) or magnetic resonance (xe2x80x9cMRxe2x80x9d) imaging are unable to distinguish metastatic prostate cancer involvement of lymph nodes by criterion other than size (i.e.,  greater than 1 cm). Therefore, by definition, these imaging modalities are inherently insensitive in the detection of small volume ( less than 1 cm) disease as well as non-specific in the detection of larger volume adenopathy. A recent study assessed the accuracy of MR in patients with clinically localized prostate cancer (Rifkin et al., N. Engl. J. Med., 323:621-626 (1990)). In this study, 194 patients underwent a MRI examination and 185 of these patients had a lymph node dissection. Twenty-three (13%) of the patients had pathologically involved lymph nodes. MRI was suspicious in only 1 of these 23 cases, resulting in a sensitivity of 4%. Similar results have also been noted with CT scans (Gasser et al., N. Engl. J. Med. (Correspondence), 324(7):49-495 (1991)).
The elevation of serum acid phosphatase activity in patients having metastasized prostate carcinoma was first reported by Gutman et al., J. Clin. Invest., 17:473 (1938). In cancer of the prostate, prostatic acid phosphatase is released from the cancer tissue into the blood stream with the result that the total serum acid phosphatase level can be greatly increased above normal values. Numerous studies of this enzyme and its relation to prostatic cancer have been made since that time, e.g. Yan, Amer. J. Med., 56:604 (1974). However, the measurement of serum acid phosphatase is elevated in about 65-90 percent of patients having carcinoma of the prostate with bone metastasis; in about 30 percent of patients without roentgenological evidence of bone metastasis; and in about only 5-10 percent of patients lacking clinically demonstrable metastasis.
Prior art attempts to develop a specific test for prostatic acid phosphatase have met with only limited success, because techniques which rely on enzyme activity on a so-called xe2x80x9cspecificxe2x80x9d substrate cannot take into account other biochemical and immunochemical differences among the many acid phosphatases which are unrelated to enzyme activity of prostate origin. In the case of isoenzymes, i.e., genetically defined enzymes having the same characteristic enzyme activity and a similar molecular structure but differing in amino acid sequences and/or content and, therefore, immunochemically distinguishable, it would appear inherently impossible to distinguish different isoenzyme forms merely by the choice of a particular substrate. It is, therefore, not surprising that none of these prior art methods is highly specific for the direct determination of prostatic acid phosphatase activity (Cancer 5:236 (1952); J. Lab. Clin. Med., 82:486 (1973); Clin. Chem. Acta, 44:21 (1973); and J. Physiol. Chem., 356:1775 (1975)).
In addition to the aforementioned problems of non-specificity, which appear to be inherent in many of the prior art reagents employed for the detection of prostate cancer phosphatase, there have been reports of elevated serum acid phosphatase associated with other diseases, which further complicates the problem of obtaining an accurate clinical diagnosis of prostatic cancer. For example, Tuchman et al. noted that serum acid phosphatase levels appear to be elevated in patients with Gaucher""s disease (Tuchman et al., Am. J. Med., 27:959 (1959)).
Due to the inherent difficulties in developing a xe2x80x9cspecificxe2x80x9d substrate for prostate acid phosphatase, several researches have developed immunochemical methods for the detection of prostate acid phosphatase. However, the previously reported immunochemical methods have drawbacks of their own which have precluded their widespread acceptance. For example, Shulman et al. described an immunodiffusion test for the detection of human prostate acid phosphatase (Shulman et al., Immunology, 93:474 (1964)). Using antisera prepared from a prostatic fluid antigen obtained by rectal massage from patients with prostatic disease, no cross-reactivity precipitin line was observed in the double diffusion technique against extracts of normal kidney, testicle, liver, and lung. However, this method has the disadvantages of limited sensitivity, even with the large amounts of antigen employed, and of employing antisera which may cross-react with other, antigenically unrelated serum protein components present in prostatic fluid.
WO 79/00475 to Chu et al. describes a method for the detection of prostatic acid phosphatase isoenzyme patterns associated with prostatic cancer which obviates many of the above drawbacks. However, practical problems are posed by this method, such as the need for a source of cancerous prostate tissue from which the diagnostically relevant prostatic acid phosphatase isoenzyme patterns associated with prostatic cancer are extracted for the preparation of antibodies thereto.
In recent years, considerable effort has been spent to identify enzyme or antigen markers for various types of malignancies with the view towards developing specific diagnostic reagents. Previous investigators have demonstrated the occurrence of human prostate tissue-specific antigens. The ideal tumor marker would exhibit, among other characteristics, tissue or cell-type specificity.
Theoretically, radiolabeled monoclonal antibodies (xe2x80x9cmAbsxe2x80x9d) offer the potential to enhance both the sensitivity and specificity of detecting prostatic cancer within lymph nodes and elsewhere. While many mAbs have previously been prepared against prostate related antigens, none of these mAbs were specifically generated with an imaging objective in mind. Nevertheless, the clinical need has led to evaluation of some of these mAbs as possible imaging agents (Vihko et al., Biotechnology in Diagnostics, 131-134 (1985); Babian et al., J. Urol., 137:439-443 (1987); Leroy et al., Cancer, 64:1-5 (1989); Meyers et al., The Prostate, 14:209-220 (1989)).
In some cases, the monoclonal antibodies developed for detection and/or treatment of prostate cancer recognize antigens specific to malignant prostatic tissues. Such antibodies are thus used to distinguish malignant prostatic tissue (for treatment or detection) from benign prostatic tissue. See U.S. Pat. No. 4,970,299 to Bazinet et al. and U.S. Pat. No. 4,902,615 to Freeman et al.
Other monoclonal antibodies react with surface antigens on all prostate epithelial cells whether cancerous or benign. See U.S. Pat. Nos. 4,446,122 and Re 33,405 to Chu et al., U.S. Pat. No. 4,863,851 to McEwan et al., and U.S. Pat. No. 5,055,404 to Ueda et al. However, the antigens detected by these monoclonal antibodies are present in the blood and, therefore, compete with antigens at tumor sites for the monoclonal antibodies. This causes background noise which makes the use of such antibodies inadequate for in vivo imaging. Furthermore, such antibodies, if bound to a cytotoxic agent, could be harmful to other organs if used in therapy.
Horoszewicz et al., Anticancer Research, 7:927-936 (1987) (hereinafter xe2x80x9cHoroszewiczxe2x80x9d) and U.S. Pat. No. 5,162,504 to Horoszewicz describe an antibody, designated 7E11, which recognizes prostate specific membrane antigen (xe2x80x9cPSMAxe2x80x9d). Israeli et al., Cancer Research, 53:227-230 (1993) (hereinafter xe2x80x9cIsraelixe2x80x9d) describes the cloning and sequencing of PSMA and reports that PSMA is prostate-specific and shows increased expression levels in metastatic sites and in hormone-refractory states. Other studies have indicated that PSMA is more strongly expressed in prostate cancer cells relative to cells from the normal prostate or from a prostate with benign hyperplasia. Furthermore, PSMA is not found in serum (Troyer et al., Int. J. Cancer, 62:552-558 (1995)).
These characteristics make PSMA an attractive target for antibody-mediated targeting for imaging and therapy of prostate cancer. Imaging studies using indium-labeled 7E11 have indicated that the antibody localizes quite well to both the prostate and to sites of metastasis. In addition, 7E11 appears to have clearly improved sensitivity for detecting lesions compared to other currently available imaging techniques, such as CT and MR imaging or bone scan (Bander, N. H., Sem. in Oncology, 21:607-612 (1994)).
However, the use of 7E11 and other known antibodies to PSMA to mediate imaging and therapy has several disadvantages. First, PSMA is an integral membrane protein known to have a short intracellular tail and a long extracellular domain. Biochemical characterization and mapping studies (Troyer et al., Urol. Oncol., 1:29-37 (1995)) have shown that the epitope of the antigenic site to which the 7E11 antibody binds is present on the intracellular portion of the PSMA molecule. Because antibody molecules do not, under normal circumstances, cross the cell membrane unless they bind to the extracellular portion of a molecule and become translocated intracellularly, the 7E11 antibody does not have access to its antigenic target site in an otherwise healthy, viable cell.
Consequently, imaging using 7E11 is limited to the detection of dead cells within tumor deposits. What is needed is a method to separate living, viable prostatic cells from tissues or fluids to enhance the detection of malignant prostatic cells.
Although the inadequacies and problems in the diagnosis and treatment of one particular type of cancer are the focus of the preceding discussion, prostate cancer is merely a representative model. The diagnosis and treatment of numerous other cancers have similar problems. Therefore, a method to enhance the separation of malignant cells from biological fluids that is applicable to a wide variety of cancers would be even more desirable.
An antibody against the prostate-specific antigen (xe2x80x9cPSAxe2x80x9d) has been used to select prostate cells. However, due to the absence of PSA from cell surface membranes, and due to the large concentration of PSA protein in semen, the antibody to PSA is known to stain nonepithelial cells in addition to the cytokeratin-positive populations (Barren, III, R. J. et al., Prostate, 36:181-188 (1998)). Moreover, whereas PSA protein is known to be more highly expressed by differentiated prostatic epithelial cells, PSMA protein is highly expressed in undifferentiated prostatic epithelial cells and tumors (Bostwick, D. G. et al., Cancer, 82:2256-2261 (1998)).
Copending U.S. patent applications Ser. Nos. 08/838,682 now U.S. Pat. No. 6,107,090, and 08/895,914 now U.S. Pat. No. 6,136,311, which are hereby incorporated in their entirety, describe procedures for detecting and ablating or killing normal, benign hyperplastic, and cancerous prostate epithelial cells. The method employs a biological agent, such as an antibody or a binding portion of an antibody, to bind an extracellular domain of the PSMA protein on such cells. The biological agent is contacted with the cells under conditions that allow both binding of the biological agent to the PMSA protein on the cells for detecting and ablating or killing of the cells. The biological agent may be used alone or in combination with a substance effective to kill the cells. The biological agent may also be used to detect normal, benign hyperplastic, or cancerous prostate epithelial cells, or portions thereof, in a biological sample.
Previously, a modified ficoll-hypaque separation procedure and sorting by flow cytometry was used to separate prostatic epithelial cells from semen. By ficoll-hypaque gradient separation, separation of intentionally added cancer cells from fresh semen was achieved but the recovery rate was highly variable. By flow cytometry this cell separation based on size and granularity identified by light scatter characteristics was accomplished. The fact that the target cell population could be isolated without requiring the use of fluorescent dyes (which may have interfered with subsequent assays) was significant. However, the recovery of prostate epithelial cells was not consistent (unpublished observation).
The present invention therefore, provides an improved method to isolate normal, benign hyperplastic, or cancerous prostate epithelial cells from semen.
The present invention provides a method for the isolation of epithelial cells from a solution, comprising providing a biologic agent capable of binding to an extracellular domain of prostate specific membrane antigen (PSMA), contacting the biologic agent with a magnetizable medium under conditions permitting binding of the biologic agent to the magnetizable medium, contacting a solution containing the epithelial cells with the magnetizable medium under conditions permitting binding of the biologic agent to the epithelial cells to form a complex including the magnetizable medium, the biologic agent, and the epithelial cells, contacting the complex with a magnetized matrix under conditions permitting removal of the complex from the solution, and eluting the epithelial cells from the magnetized matrix. The biologic agent may be a polyclonal antibody, a monoclonal antibody, or a portion of a monoclonal antibody, such as an F(ab) fragment, an F(abxe2x80x2)2 fragment, or an Fv fragment. The biologic agent may also be a probe or a ligand capable of binding to PMSA. The biologic agent is preferably a monoclonal antibody or a portion thereof that binds specifically to the extracellular domain of PSMA, such as mAb E99, mAb J415, mAb J533, or mAb J591. The biologic agent is more preferably a monoclonal antibody or a portion thereof of mAb J591. The monoclonal antibodies, polyclonal antibodies, and portions thereof may be produced by methods that are well known to those in the art.
The present invention also provides a method for detecting the presence of cancerous vascular endothelial cells, comprising providing a biologic agent capable of binding to an extracellular domain of prostate specific membrane antigen (PSMA), contacting the biologic agent with a magnetizable medium under conditions permitting binding of the biologic agent to the magnetizable medium, contacting a solution containing the cancerous vascular endothelial cells with the magnetizable medium bound to the biologic agent under conditions permitting binding of the biologic agent to the cancerous vascular endothelial cells to form a complex containing the magnetizable medium, the biologic agent, and the cancerous vascular endothelial cells, contacting the complex with a magnetized matrix under conditions permitting isolation of the complex from the solution, eluting the epithelial cells from the magnetized matrix, and detecting the presence of the cancerous vascular endothelial cells.
The epithelial cells in the solution may be normal epithelial cells, benign hyperplastic epithelial cells, cancerous epithelial cells, normal prostate epithelial cells, benign hyperplastic prostate epithelial cells, or cancerous prostate epithelial cells. The method is particularly applicable to the isolation of cancerous prostate epithelial cells, such as prostatic adenocarcinoma cells.
The epithelial or vascular endothelial cells may be contained in any appropriate fluid, including a biological fluid or a tissue culture fluid. The biological fluid may be blood, urine, semen, seminal fluid, lymph, cerebrospinal fluid, mucus, tears, sweat, gastric fluid, saliva, synovial fluid, or a bone marrow suspension.